Oxygen sensors

ABSTRACT

Oxygen sensing luminescent dyes, polymers and sensors comprising these sensors and methods of using these sensors and systems are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/830,798, filed Dec. 4, 2017, which is a continuation of U.S. patentapplication Ser. No. 15/189,448, now U.S. Pat. No. 9,867,560, filed Jun.22, 2016, which is a continuation of U.S. patent application Ser. No.14/209,252, now U.S. Pat. No. 9,375,494, filed Mar. 13, 2014, whichclaims the benefit of U.S. provisional patent application 61/784,925filed on Mar. 14, 2013, titled “Oxygen Sensors”, the disclosure of eachof which is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

The present disclosure is in the field of luminescent dyes, polymers andbiosensors.

BACKGROUND

Diagnosis, treatment and management of some medical conditions requiremonitoring of oxygen concentration in the afflicted organ or tissue. Forexample, Peripheral Arterial Disease (PAD), a disease that ischaracterized by plaque buildup in arteries that carry blood to theextremities, head, or organs, if left untreated, can lead to completeblockage of lower extremity arteries and requires either open bypasssurgery or endovascular intervention. Annually, at least 140,000 suchrevascularization procedures are conducted in the US alone to restoreblood flow to ischemic tissues. Thus, ensuring that blood and oxygenflow are adequately restored and maintained during and after therevascularization technique is highly desirable. Current monitoringmethods are expensive, cumbersome, time consuming, and do not provideaccurate, continuous tissue oxygenation information. Thus, there isclearly a need for a better long-term oxygen tissue monitoring system.Doing so non-invasively with minimal user maintenance is essential, andsensor longevity of days to months is crucial in actual userenvironments.

Such real-time, continuous measurement of oxygen concentration (partialpressure) in tissues can be achieved by the use of sensors inserted orimplanted into the tissue and measuring the signal generated by thesensor by a device located outside the body. Luminescence provides auseful tool for the design of such sensors. Luminescent oxygen sensorsare based on the phenomenon that oxygen has a quenching effect on themolecular luminescence of various chemical compounds and that thiseffect can be employed for measuring oxygen concentrations (partialpressure) in vivo. The sensors, which are monitored optically throughthe skin, require a highly stable dye with excitation and emissionspectra in the near-infrared (NIR) optical window of the skin. These dyeproperties are crucial for the successful design of a luminescent oxygensensor that can be implanted deep into tissue. Monitoring non-invasivelythrough the skin requires the use of dyes with excitation and emissionwavelengths in the optical window of the skin (approximately 550 to 1000nm) to minimize light scattering and absorbance, and achieve a highsignal-to-noise ratio. However, commercially available NIR dyes can beprone to photobleaching. Palladium porphyrins, such astetracarboxyphenyl porphyrin (Pd-TCPP) have a very large Stokes shiftand emission in the NIR. However, they unfortunately require excitationwith green light (525 nm), which is largely absorbed by the skin and theunderlying tissue. Additionally, currently available sensors, made ofrigid materials that vastly differ from the mechanical properties oftissue in which they are implanted, are bulky and inconvenient, andinduce a series of biological events upon implantation that ultimatelyculminate in the formation of a fibrous capsule that walls it off fromthe body.

Thus, until the present invention there remains a clear need in the artto provide improved stable, near-IR luminescent compounds and sensorsfor direct, rapid and accurate measurement of oxygen levels in tissue,particularly in vivo.

SUMMARY

Disclosed herein are luminescent dyes, polymers comprising said dyes,and sensors comprising the polymers of the present invention.

In one embodiment, the present invention relates to a compound ofFormula 1:

wherein:

-   -   M is H, Pd, Zn, Pt, Gd or Yb;    -   each R¹ is same or different and independently        C(O)X—(CH₂)_(n)—YC(O)C(R⁴)CH₂,        C(O)X—(CH₂Cl₂O)_(m)CH₂CH₂—YC(O)C(R⁴)CH₂ or COOH;    -   R⁷ is C(O)X—(CH₂)_(n)—YC(O)C(R⁴)CH₂ or        C(O)X—(CH₂CH₂O)_(m)CH₂CH₂—YC(O)C(R⁴)CH₂;    -   R² and R³ are hydrogen or are fused, in each case, to form a        cycloalkenyl, aryl, or heteroaryl group;    -   X is O or NR⁵;    -   Y is O or NH;    -   R⁵ and R⁴ are independently H or C1-C4 alkyl;    -   each R⁶ is the same or different and independently H or F;    -   n is 1-10; and    -   m is 1-300.

In another aspect, the present invention relates to a polymer comprisingas a monomer repeat unit, the residue of the compound of Formula 1. Thepolymers provided herein can be luminescent biocompatible hydrogels.

In further embodiments, the present invention relates to variousluminescent sensors comprising the polymers provided herein fordetecting an analyte, e.g., oxygen, in vivo or in vitro. The sensors canbe in the form of a powder, fabric (e.g., wound dressing), sutures,needle, rod, disk or any other suitable form.

In another aspect, the luminescent sensors provided herein aretissue-integrating or comprise a tissue-integrating scaffold and producea detectable signal in the presence of the analyte; and further whereinthe sensors provide detection of the analyte when placed (e.g.,implanted) into the tissue of a subject. The tissue-integrating sensorsas described herein can provide long-term detection of the analyte(s).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts Compound 2 (Pd-BP) absorption and emission spectra.Spectra were taken of covalently bound Pd-BP in pHEMA hydrogel.Excitation at 633 nm gave 805 nm emission, confirming shift into theNIR.

FIG. 2 demonstrates that Compound 2 (Pd-BP) incorporated into a pHEMAhydrogel sensor enables brighter signals from deeper within the tissue.Images above show intensity of NIR Pd-BP (A) and green-ex Pd-TCPP (B)subcutaneous hydrogel implants measured in a rat carcass. Pd—BP issignificantly brighter than TCPP due to the NIR excitation and emissionwavelengths, which allow much greater light penetration into the skin,enabling deeper sensor placement.

FIG. 3 depicts luminescence signal of pHEMA O₂ sensor implanted in amouse brain.

FIG. 4 depicts luminescence of oxygen sensors implanted in rat skin (170days). Intensity varies as a function of implantation depth (datanormalized to baseline fluorescence) and tissue oxygen concentration.Inhaled oxygen was modulated between 100% and 12% and images werecollected every 30 s in a Caliper IVIS (Ex=640 nm, Em=: 800 nm). Regionsof interest (ROIs) were drawn around the sensors and the data plottedversus time. Data is shown in FIG. 6.

FIG. 5 shows a SEM image of tissue-integrating porous hydrogel scaffold.

FIG. 6 demonstrates determination of photostability Pd-BP. Gels in PBS(pH 7.4, 37° C.) were illuminated using a 525 nm LED at a 40% dutycycle. Both lifetime signal remains constant.

FIG. 7A depicts that the response of the porous, tissue-integratedsensors is rapid (30 seconds). The solid sensors are rod-shaped andmaterial composition.

FIG. 7B depicts that the response of the solid sensor response is muchslower (plateau not even reached after 5 minutes). The solid sensors arethe same rod-shape and material composition as shown in FIG. 7A.

FIG. 8A depicts dynamic response of Pd-BP hydrogels to 02. The responseis linear with good sensitivity and rapid response time.

FIG. 8B depicts a Stern-Vomer plot of O₂ quenching efficiency with Pd-BP(B).

FIG. 9A depicts dynamic response of GOx/Pd-BP gel to glucose.

FIG. 9B depicts a normalized glucose dose-response curve.

FIG. 10A depicts detectable modulating sensor signal from the O₂ sensor.

FIG. 10B depicts histological analysis of pig biopsy containing thesensor.

FIG. 11A depicts solid sensor response to deoxygenation (0.12 FIO2) andre-oxygenation (1.00 FIO2).

FIG. 11B shows fluorescent micrographs of solid sensors and surroundingtissue samples at 7 and 28 days after implantation.

FIG. 11C depicts porous, tissue-integrating sensor response todeoxygenation (0.12 FIO2) and re-oxygenation (1.00 FIO2).

FIG. 11D shows fluorescent micrographs of porous, tissue-integratingsensors and surrounding tissue samples at 7 and 28 days afterimplantation.

DETAILED DESCRIPTION

Described herein are polymerizable luminescent dyes useful forincorporation into polymers and polymers comprising as monomeric unitsresidues of the dyes of the present invention. The dyes and the polymersare useful, for example, in sensing and imaging applications, forexample, accurate and optionally long term measurements of oxygen invivo and in vitro.

Additionally, described herein are sensors comprising the polymers ofthe present invention. The sensors can be implanted into a tissue of asubject and used for long-term or short-term continuous andsemi-continuous collection of data of various biochemical analytes,optionally without the use of implantable hardware of any type and/orenzymatic and electrochemical detection methods. In one aspect, thesensors are tissue integrating, e.g., allow capillaries to grow in closeproximity to all regions of the sensor (e.g., on the surface andinside), which results in accurate analyte measurements, including overlong term. In another aspect, in addition to the luminescent dyes and/orthe polymers of the present invention, the sensors comprise an oxidase,such as, but not limited to, glucose oxidase, and the luminescent dyesand/or their residues incorporated as monomeric units into the polymersmeasure the consumption of oxygen by the oxidase, thus, the sensors canprovide detection of a number of analytes other than oxygen, such as,but not limited to, glucose.

Advantages of the dyes and luminescent polymers provided herein include,but are not limited to: (1) excitation and emission wavelengths in theoptical window of the skin (approximately 550 nm to 1000 nm) allowingdetection of analytes deep within a tissue or an organ; (2) highsignal-to-noise ratio; (3) large Stokes shifts and emission; (4)photostability, e.g., the dyes and/or polymers do not undergo rapidphotobleaching.

Advantages of the sensors described herein include, but are not limitedto: (1) providing devices that generate stable signal over a long periodof time (e.g., greater than a week, greater than a month, greater than 6months), (2) providing devices that are placed or implanted andintegrate into the subject's tissue (e.g., through tissue and/orcapillary in-growth); (3) providing devices which can be implantedthrough syringe injection or trocar injection, meaning that no surgeryis required to put the sensing media in place in the body; (4) providingdevices that do not include sensor electronics in the body; (5)providing devices that accurately assess analyte (e.g., oxygen)concentration for long periods of time (e.g., greater than a week,typically weeks, months or years) and/or (6) providing devices of smalldimensions which will give result in increased patent comfort and betteracceptance by the body.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an”, and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to a sensor comprising “a sensing moiety” includes devicescomprising of two or more sensing moieties. Likewise, reference to “ananalyte” refers to two or more analytes.

Definitions

The term “tissue integrating” refers to a material (e.g., scaffold)which, when integrated into living tissue remains in close proximitywith the blood vessels of the tissue (e.g., capillaries).

By “long-term” is meant that the implant senses the analyte for greaterthan about 7 days, for example weeks, months, or years.

By “biodegradable” or “bioabsorbable” is meant that the material iscapable of being broken down by the subject's body over a period oftime, ranging from days to weeks to months or years.

By “hydrogel” is meant a material that absorbs a solvent (e.g. water),undergoes rapid swelling without discernible dissolution, and maintainsthree-dimensional networks capable of reversible deformation.

The term “stimuli-responsive” refers to substances, e.g., polymers, thatchange their physical state, e.g., undergo a phase transition, whenexposed to an external stimulus or according to the environment they arein. Non-limiting examples of such polymers are “smart polymers” (KumarA. et al., Smart polymers: Physical forms and bioengineeringapplications. Prog. Polym. Sci. 32 (2007) 1205-1237).

A. Luminescent NIR Dyes

In one aspect, this invention provides a compound of Formula 1:

wherein

M is H, Pd, Zn, Pt, Gd or Yb;

each R¹ is same or different and independentlyC(O)X—(CH₂)_(n)—YC(O)C(R⁴)CH₂, C(O)X—(CH₂CH₂O)_(m)CH₂CH₂—YC(O)C(R⁴)CH₂or COOH;

R⁷ is C(O)X—(CH2)_(n)—YC(O)C(R⁴)CH₂ or C(O)X—(CH2CH2O)_(m)CH2CH2-YC(O)C(R4)CH2;

R² and R³ are hydrogen or are fused, in each case, to form acycloalkenyl, aryl, or heteroaryl group;

X is O or NR⁵;

Y is O or NH;

R⁵ and R⁴ are independently H or C1-C4 alkyl;

each R⁶ is same or different and, independently, H or F;

n is 1-10; and

m is 1-300.

In one embodiment, M is Pd. In another embodiment, R¹ and R⁷ are bothC(O)NH(CH₂)₂OC(O)C(CH₃)CH₂. In another embodiment, R₁ isC(O)NH(CH₂)₂OC(O)C(CH₃)CH₂ and R⁷ is COOH. In yet another embodiment,two of the R¹ are C(O)NH(CH₂)₂OC(O)C(CH₃)CH₂, one of the R¹ is COOH andR⁷ is COOH. In another embodiment, one of the R¹ isC(O)NH(CH₂)₂OC(O)C(CH₃)CH₂, two of the R¹ are COOH, and R⁷ is COOH. Inone embodiment, all R¹ and R⁷ are COOH.

In another embodiment, R¹ and R⁷ are bothC(O)X—(CH₂CH₂O)_(m)CH₂CH₂—YC(O)C(R₄)CH₂. In another embodiment, R¹ isC(O)X—(CH₂CH₂O)_(m) CH₂CH₂—YC(O)C(R₄)CH₂ and R⁷ is COOH. In yet anotherembodiment, two of the R¹ are C(O)X—(CH₂CH₂O)_(m) CH₂CH₂—YC(O)C(R₄)CH₂,one of the R¹ is COOH and R⁷ is COOH. In another embodiment, one of theR¹ is C(O)X—(CH₂CH₂O)_(m) CH₂CH₂—YC(O)C(R₄)CH₂, two of the R¹ are COOH,and R⁷ is COOH. In one embodiment, all R¹ and R⁷ are COOH.

In another embodiment, R¹ and R⁷ are both C(O)X—(CH₂)_(n)—YC(O)C(R⁴)CH₂.In another embodiment, R¹ is C(O)X—(CH₂)_(n)—YC(O)C(R⁴)CH₂ and R₇ isCOOH. In yet another embodiment, two of the R₁ areC(O)X—(CH₂)_(n)—YC(O)C(R⁴)CH₂, one of the R¹ is COOH and R⁷ is COOH. Inanother embodiment, one of the R₁ is C(O)X—(CH₂)_(n)—YC(O)C(R⁴)CH₂, twoof the R¹ are COOH, and R⁷ is COOH. In one embodiment, all R¹ and R⁷ areCOOH.

In one embodiment, R² and R³ are fused to form a heteroaryl group. Inone embodiment, R² and R³ are fused to form a cycloalkenyl group. In oneembodiment, R² and R³ are fused to form a tetracyclohexeno group. In oneembodiment, R² and R³ are fused to form an aryl group. In oneembodiment, the aryl group is perfluorinated. In one embodiment, R² andR³ are fused to form a benzo group. In another embodiment, R² and R³ arefused to form a naphtho group.

In one embodiment, R¹ comprises an oligoethyene glycol linker having2-300 ethylene units. In another embodiment, R⁷ comprises anoligoethyene glycol linker having 2-300 ethylene units.

In one specific embodiment, M is Pd, R¹ and R⁷ are bothC(O)NH(CH₂)₂OC(O)C(CH₃)CH₂, and R2 and R3 are H.

In one specific embodiment, M is Pd, R¹ and R⁷ are bothC(O)NH(CH₂)₂OC(O)C(CH₃)CH₂, and R² and R³ are fused to form a benzenering.

In one embodiment, the compound of Formula 1 is a near-IR luminescentdye. In one embodiment, the compound of Formula 1 has an absorptionmaximum between 500 nm and 800 nm. In one specific embodiment, thecompound of Formula 1 has an absorption maximum between 500 nm and 700nm. In one embodiment, the compound of Formula 1 has an emission maximumbetween 500 and 1000 nm. In one embodiment, the compound of Formula 1has an emission maximum between 650 and 900 nm. In one specificembodiment, the compound of Formula 1 has an emission maximum between800 and 900 nm. In one embodiment, the compound of Formula 1 of thepresent invention is photostable and has excitation and emission spectrain the NIR optical window of the skin.

For example, in a preferred embodiment, as illustrated by FIG. 1, theCompound 2 of Formula 2 has an absorption maximum at 633 nm and anemission maximum at 805 nm when co-polymerized with HEMA into ahydrogel.

In some embodiments, the dyes of the present invention are encapsulatedinto a solid, oxygen-impermeable nanosphere. The nanospheres can be usedfor luminescent, non-oxygen sensitive applications.

B. Polymers

The fluorescent dyes of the present invention comprise polymerizablegroups, e.g., residue of acrylic or methacrylic acid, and can beco-polymerized with other monomers to provide polymers comprisingnear-IR luminescent groups. When the compounds have 2 or morepolymerizable groups, the polymers obtained from their co-polymerizationwith other monomers can be crosslinked. Alternatively, anothercrosslinking monomer can be added into the polymerization mixture toachieve a higher degree of crosslinking of the resulting polymer.

Polymers described herein can be prepared in any suitable manner.Suitable synthetic methods used to produce the polymers provided hereininclude, by way of non-limiting example, cationic, anionic and freeradical polymerization. In certain embodiments, polymer synthesis isperformed neat or in any suitable solvent. Suitable solvents include,but are not limited to, pentane, hexane, dichloromethane, chloroform,water, ethylene glycol, propylene glycol, DMSO or dimethyl formamide(DMF). In certain embodiments, the polymer synthesis is performed at anysuitable reaction temperature, including, e.g., from about −50° C. toabout 100° C., or from about 0° C. to about 70° C.

Preferably the polymers are prepared by the means of a free radicalpolymerization. When a free radical polymerization process is used, (i)the monomer, (ii) optionally, the co-monomer(s), and (iii) an optionalsource of free radicals are provided to trigger a free radicalpolymerization process. In some embodiments, the source of free radicalsis optional because some monomers may self-initiate upon heating at hightemperature. In certain instances, after forming the polymerizationmixture, the mixture is subjected to polymerization conditions. Suchconditions are optionally varied to any suitable level and include, byway of non-limiting example, temperature, pressure, light, atmosphere,ratios of starting components used in the polymerization mixture andreaction time. The polymerization is carried out in any suitable manner,including, e.g., in solution, dispersion, suspension, emulsion or bulk.

In some embodiments, initiators are present in the reaction mixture. Anysuitable initiator is optionally utilized if useful in thepolymerization processes described herein. Such initiators include, byway of non-limiting example, one or more of alkyl peroxides, substitutedalkyl peroxides, aryl peroxides, substituted aryl peroxides, acylperoxides, alkyl hydroperoxides, substituted alkyl hydroperoxides, arylhydroperoxides, substituted aryl hydroperoxides, heteroalkyl peroxides,substituted heteroalkyl peroxides, heteroalkyl hydroperoxides,substituted heteroalkyl hydroperoxides, heteroaryl peroxides,substituted heteroaryl peroxides, heteroaryl hydroperoxides, substitutedheteroaryl hydroperoxides, alkyl peresters, substituted alkyl peresters,aryl peresters, substituted aryl peresters, or azo compounds. Inspecific embodiments, benzoylperoxide (BPO) and/or AIBN are used asinitiators.

In some embodiments, polymerization processes are carried out in acontrolled (living) mode. Preferred controlled (living) polymerizationprocesses include reversible addition-fragmentation chain transfer(RAFT) polymerization processes and Atom Transfer Radical Polymerization(ATRP).

In certain embodiments, the polymer of the present invention is ahydrogel. For example, the hydrogel can be prepared by reactinghydroxyethyl methacrylate (HEMA), to form poly(hydroxyethylmethacrylate), pHEMA. Furthermore, various comonomers can be used incombination to alter the hydrophilicity, mechanical and swellingproperties of the hydrogel (e.g. PEG, NVP, MAA). Non-limiting examplesof polymers include 2-Hydroxyethyl methacrylate, polyacrylamide,N-vinylpyrrolidone, N,N-Dimethylacrylamide, poly(ethylene glycol)monomethacrylate (of varying molecular weights), diethylene glycolmethacrylate, N-(2-hydroxypropyl)methacrylamide, glycerolmonomethacrylate, 2,3-dihydroxypropyl methacrylate and combinationsthereof. Non-limiting examples of cross-linkers include tetraethyleneglycol dimethacrylate, poly(ethylene glycol) (n) diacrylate (of varyingmolecular weights), ethoxylated trimethylolpropane triacrylate,bisacrylamide and combinations thereof. Non-limiting examples ofinitiators include Irgacure Series (UV), Azobisisobutyronitrile (AIBN)(thermal), Ammonium Persulfate (APS) (thermal).

In a specific embodiment, the polymer is a luminescent hydrogel preparedby co-polymerization of HEMA and compound of Formula 1. In a preferredembodiment, the hydrogel is prepared by co-polymerization of variousmolar amounts of compound of Formula 2 mixed with 2-hydroxyethylmethacrylate (HEMA) monomer, tetraethylene glycol dimethacrylate(TEGDMA) crosslinker, Irgacure 651 initiator, water and co-solvent,followed by UV-initiated polymerization. In another embodiment, thepolymer contains 1 mM final concentration of Compound of Formula 1. In aspecific embodiment, the polymer is an oxygen sensingpoly(2-hydroxyethyl methacrylate) (pHEMA) scaffold prepared byco-polymerization of HEMA (2-hydroxyethyl methacrylate) (50 Wt %),TEGDMA (triethyleneglycol-dimethacrylate) (1 Wt % 1), ethylene glycol(20 Wt %), water (25.5 Wt %), the photoinitiator Irgacure 651 (0.5%vol/vol) and 3% of Compound 2.

The polymer of the present invention may be degradable, either by thebody (biodegradable) or by the application of an external initiator tostart or speed up the degradation process (e.g. UV, ultrasonics, radiofrequency, temperature, or other exogenous sources to initiatedegradation). For example, the polymer may be biodegradable orbioresorbable or may comprised any biodegradable or bioresorbablesegments, including but not limited to degradable forms of alginates,poly(lactic acid), poly(vinyl alcohol), polyanhydrides, poly(glycolicacid), microporous polyesters, microporous polyethers and cross-linkedcollagen. One specific example is UV-photopolymerization ofpoly(ethylene glycol)-diacrylate and acrylated protease-degradablepeptides and VEGF as described by Phelps, et al (2010) Proc. Nat'l.Acad. Sci. USA 107(8):3323-3328.

In one embodiment, polymers provided herein are biocompatible. Inanother aspect of the invention, the polymers are biodegradable.Degradable hydrogels can be synthesized using Atom Transfer RadicalPolymerization (ATRP) through co-polymerization of the HEMA withpolymerizable luminescent dyes of the present invention. Porous sensorscaffolds, based on non-degradable and degradable oxygen-sensinghydrogels, can be generated by using a sphere-templating fabricationtechnique. Degradable and non-degradable HEMA reagents and polymerizabledye will be polymerized over templating microspheres, which aresubsequently dissolved away with solvent to generate desirablenon-degradable and degradable scaffolds. Briefly, using controlled ATRP,HEMA will be polymerized in the presence of bi-functional degradablePCL-based ATRP initiator and cross-linker. In this synthesis scheme,pHEMA chains grow at the same rate from both sides of degradableinitiator, resulting in degradation products with a MW that is half thatof the parent polymer. By controlling the MW of the parent polymer andthe PEG and PCL units in the initiator and/or crosslinker, thedegradation rate of the polymers can be varied. Limiting the MW of theparent polymer to 10 kDa results in degradation products that can becleared by the body and an increased degradation rate while stillpreserving the hydrogel's mechanical strength.

In certain embodiments the polymers provided herein arestimuli-responsive, e.g., temperature or pH-sensitive polymers. Onenon-limiting example of such a stimuli-responsive polymer is atemperature-sensitive polymer derived from co-polymerization of NIPAM.Such polymers are useful for implantation of the sensor comprising saidpolymers in a desired location within tissue by first dissolving thepolymer in a suitable for injection media at a lower than bodytemperature and then injecting the resulting solution into the tissueand/or at desired location of the body. As the polymer is subjected to ahigher (e.g., body) temperature, it precipitates in or near the site ofthe injection where monitoring of oxygen is required.

C. Sensors

In some embodiments, the polymer of the present invention isincorporated into a sensor useful for detection of an analyte. Thedetection of the analyte can be in vitro or in vivo. The remainingsentences of this paragraph describe how the word “polymer” is used insection titled “C. Sensors”. The polymer may have the molecules ofFormula 1 and/or Formula 2 covalently bound to the polymer backbone. Themolecules of Formula 1 and/or Formula 2 maybe attached to (e.g. via acovalent bond or other means) or contained within nanoparticle carriersor microparticle carriers or other carriers that are attached to orcontained within the polymer. Such carriers may be covalently bound tothe polymer backbone. The word polymer can be used interchangeably withthe word sensor.

In one non-limiting example, the polymer is incorporated into anoxygen-sensing wound dressing that can be used to monitor the process ofwound healing, e.g. to constantly and non-invasively assess one of thecritical factors of healing (i.e. oxygenation).

In another embodiment, the polymer is incorporated into a powder, whichis used directly in the wound as a sensor for wound-healing monitoring.The sensor of the present invention can also be in the form of aninjectable, implant, a mesh or sutures to be used in applications whichbenefit from monitoring of oxygenation of skin or the underlying tissue,including, but not limited to wound healing monitoring, skin closure,hernia repair, flap transfer surgeries, reconstructive surgery, andother plastic surgery applications. The sensor of the present inventioncan also be used for measurement for microcirculatory dysfunction andperipheral artery disease. Specifically in re-vascularization proceduresor upon administration of drug, tissue oxygen may be directly monitored.The sensor of the present invention can also be used in oncologyapplications to determine the degree of hypoxia in a tissue or an organ.In one embodiment, the sensor is used to monitor tumor growth in animal,including but not limited to, mouse or rat models used in oncologypharmaceutical and diagnostic research and discovery, e.g., cancertherapy dosing or monitoring of tumor metabolism. The sensor of thepresent invention can also be used in monitoring the state of pulmonaryfunction, for example in COPD and asthma disease states. In yet anotherembodiment, the sensor is used for exercise or training optimization,e.g., soldier and athlete performance or personal exercise programs. Thesensor can also be in the form of an oxygen-sensing tattoo.

Yet in another embodiment, the sensors of the present invention are usedin neuroscience monitoring applications, where currently there are notools available for continuous monitoring of oxygen, for example, insubarachnoid hemorrhage monitoring.

In one embodiment, the sensor of the present invention is a solidmaterial that could be in form of a slab, rod, cylinder, particle orpowder. In a specific embodiment, the sensor is in the form of a rod. Inanother embodiment, the sensor is in the form of a cylinder.

In another embodiment, the polymer of the present invention isincorporated into a tissue-integrating scaffold to provide atissue-integrating sensor (as described in the US patent application2012/0265034, incorporated herein by reference). The sensors describedherein typically comprise a tissue-integrating scaffold (also referredto as a matrix) material. Preferably, the tissue-integrating scaffold ofthe invention may be constructed with materials and/ormicro-architecture such that the scaffold promotes tissue-integrationand/or vascularization. For example, porous scaffolds provide tissuebiomaterial anchoring and promote in-growth throughout the pores. Theresulting “hallway” or “channel” pattern of tissue growth are healthy,space-filling masses that persist over time and promote host cellintegration. Most or all of the pores of the biomaterials describedherein are preferably interconnected (co-continuous). The co-continuouspore structure of the biomaterials promotes space-filling in-growth ofcells in the implant, which in turn limits the foreign body response andleads to long-term (greater than one week and up to years) persistenceof the implant's ability to act as a sensor. Alternative structures thatprovide tissue integrating scaffolds include fibers (e.g., 1 to 10 ormore microns in diameter, such as 5, 6, 7, 8, 9, 10 or more microns),which may be arranged in non-random or random configuration.Tissue-integrating scaffolds (in any configuration) can also be formedby multiphoton polymerization techniques. Kaehr et al. (2008) Proc.Nat'l. Acad. Sci. USA 105(26):8850-8854; Nielson et al. (2009) Small1:120-125; Kasprzak, Doctoral Dissertation, Georgia Institute ofTechnology, May 2009.

The polymer of the invention, preferably in the form of atissue-integrating scaffold, may comprise any material in combinationwith the compound of Formula 1 or Formula 2, including but not limitedto synthetic polymers, naturally-occurring substances, or mixturesthereof. Exemplary synthetic polymers include, but are not limited topolyethylene glycol (PEG), 2-hydroxyethyl methacrylate (HEMA), siliconerubber, poly([epsilon]-caprolactone) dimethylacrylate, polysulfone,(poly)methy methacrylate (PMMA), soluble Teflon-AF, (poly)ethylenetetrapthalate (PET, Dacron), Nylon, polyvinyl alcohol,polyacrylamide, polyurethane, and mixtures thereof. Exemplarynaturally-occurring materials include, but are not limited to, fibrousor globular proteins, complex carbohydrates, glycosaminoglycans,extracellular matrix, or mixtures thereof. Thus, the polymer scaffoldmay include collagens of all types, elastin, hyaluronic acid, alginicacid, desmin, versican, matricelluar proteins such as SPARC(osteonectin), osteopontin, thrombospondin 1 and 2, fibrin, fibronectin,vitronectin, albumin, chitosan etc. Natural polymers may be used as thescaffold or as an additive.

In certain embodiments, the polymer of the invention, preferably in theform of a tissue-integrating scaffold, comprises a hydrogel. Forexample, the polymer may comprise a hydrogel, for example by reactinghydroxyethyl methacrylate (HEMA), poly (hydroxyethyl methacrylate),pHEMA. Furthermore, various comonomers can be used in combination toalter the hydrophilicity, mechanical and swelling properties of thehydrogel (e.g. PEG, NVP, MAA). Non-limiting examples of polymers include2-hydroxyethyl methacrylate, polyacrylamide, N-vinylpyrrolidone,N,N-dimethylacrylamide, poly(ethylene glycol) monomethacrylate (ofvarying molecular weights), diethylene glycol methacrylate,N-(2-hydroxypropyl)methacrylamide, glycerol monomethacrylate,2,3-dihydroxypropyl methacrylate and combinations thereof. Non-limitingexamples of cross-linkers include tetraethylene glycol dimethacrylate,poly(ethylene glycol) (n) diacrylate (of varying molecular weights),ethoxylated trimethylolpropane triacrylate, bisacrylamide andcombinations thereof. Non-limiting examples of initiators includeirgacure Series (UV), Azobisisobutyronitrile (AIBN) (thermal), AmmoniumPersulfate (APS) (thermal).

The polymer of the invention, preferably in the form of atissue-integrating scaffold, may be a sphere-templated hydrogel, forinstance an inverse colloid crystal, for example as described in U.S.Patent Publication No. 2008/0075752 to Ratner, et al. or other tissueintegrating materials.

The polymer of the invention, preferably in the form of atissue-integrating scaffold, may be degradable, either by the body(biodegradable) or by the application of an external initiator to startor speed up the degradation process (e.g. UV, ultrasonics, radiofrequency, or other exogenous sources to initiate degradation). Forexample, the polymer may be comprised of any biodegradable orbioresorbable polymers, including but not limited to degradable forms ofalginates, poly(lactic acid), poly(vinyl alcohol), polyanhydrides,poly(glycolic acid), microporous polyesters, microporous polyethers andcross-linked collagen. One specific example is UV-photopolymerization ofpoly(ethylene glycol)-diacrylate and acrylated protease-degradablepeptides and VEGF as described by Phelps, et al (2010) Proc. Nat'l.Acad. Sci. USA 107(8):3323-3328.

Other specific examples are polymers described by Kloxin et al (2009)Science 324:59-63 and U.S. Pat. No. 6,013,122 whose degradation iscontrolled through exposure to exogenous energy forms, as well as byAlexeev et al. (2003) Anal. Chem. 75:2316-2323; Badylak et al. (2008)Seminars in Immunology 20:109-116; Bridges et al. (2010) 94(1):252-258;Isenhlath et al. (2007) Research 83A:915-922; Marshall et al. (2004)Polymer Preprints. American Chemical Society, Division of PolymerChemistry 45:100-101; Phelps et al. (2010) Proc Nat'l Acad Sci USA.107(8):3323-8; Ostendorf and Chichkov (2006) Two Photon Polymerization:A New Approach to MicroMachining, Photonics Spectra; Ozdemir et al.(2005) Experimental and Clinical Research, Plast. Reconstr. Surg.115:183; U.S. Patent Publication No. 20080075752; Sanders et al. (2003)Journal of Biomedical Materials Research Part A 67A(4):1181-1187;Sanders et al. (2002) Journal of Biomedical Materials Research62(2):222-227; Sanders et al. (2003) Journal of Biomedical MaterialsResearch 65(4):462-467; Sanders et al. (2005) Biomaterials 26:813-818;Sanders et al. (2005) Journal of Biomedical Materials Research Part A72(3):335-342; Sanders (2003) Journal of Biomedical Materials Research67(4):1412-1416; Sanders et al. (2000) Journal of Biomedical MaterialsResearch 52(1):231-237; and Young Min Ju et al. (2008) J Biomed MaterRes 87A:136-146.

In certain embodiments, the polymer of the invention, preferably in theform of a tissue-integrating scaffold, is constructed such that tissueresponse modifiers are released from the scaffold material to promote orenhance tissue-integration and vascularization.

In addition, the polymer of the invention, preferably in the form of atissue-integrating scaffold, may be constructed such that it hasconduits, pores or pockets that are hollow or filled with degradable,angiogenic, or other substances (e.g. stem cells). As noted above, oncein the body, the biodegradation of the material filling the conduits,pores or pockets, creates space for tissue, including capillaries tointegrate with the material. The degradable material that initiallyfills the conduits, pores, or pockets may enhance vessel growth ortissue growth within the scaffold. This architecture promotes new vesselformation and maintains healthy viable tissue within and around theimplant.

The polymer of the invention, preferably in the form of atissue-integrating scaffold, may be constructed such that it ispermeable to analytes of interest (e.g., oxygen can diffuse into atissue-integrating hydrogel scaffold and reach the sensing moieties thatare embedded within the hydrogel matrix).

The polymer of the invention, preferably in the form of atissue-integrating scaffold, can be of any suitable form, including, butnot limited to block-like (or any thickness), cube-like, disk-shaped,cylindrical, oval, round, random or non-random configurations of fibersand the like. In certain embodiments, the sensor comprises one or morefibers, which may be organized in a non-random fashion (e.g., grid,layered grid, etc.) or in a random fashion.

The polymer of the invention, preferably in the form of atissue-integrating scaffold, described herein are typically combinedwith (or made up of) sensing moieties that detect one or more analytes.In one embodiment, the sensing moiety is the residue of compound ofFormula 1 and/or 2 incorporated into the tissue-integrating scaffold.

In another embodiment, the polymer of the invention, preferably in theform of a tissue-integrating scaffold, comprises, in addition to theresidue of compound of Formula 1 and/or Formula 2, a second sensingmoiety that produces or consumes oxygen, e.g., an oxidase, and theresidue of compound of Formula 1 and/or Formula 2 is used to detect thechange in the oxygen concentration generated by the second sensingmoiety. The second sensing moiety can comprise an enzyme, for exampleglucose oxidase (GOx), which is specific for the substrate glucose. Thereaction of glucose via enzymatic interaction with glucose oxidasecauses oxygen to be proportionally consumed and converted to H₂O₂. Thereduction of O₂ in the vicinity of the enzyme can be measured by usingan O₂-sensitive fluorescent dye, such as the molecules of Formula 1 andFormula 2. These dye molecules are quenched in the presence of O₂, sothe reduction of O₂ by the action of GOx, causes an increase influorescence. The amount of fluorescence emitted from the O₂ calibrationmoieties is thus proportional to the concentration of glucose in thesensor. Oxidases besides glucose oxidase for detection of other analytesbesides glucose may include billirubin oxidase, ethanol oxidase, lactateoxidase, pyruvate oxidase, histamine oxidase or other oxidase to providespecificity to other analytes of interest.

The concentration of O₂ in the tissue can also vary physiologically,thereby changing or limiting the reaction of the oxide enzyme in thesensing moieties. Therefore, the O₂ concentration in the sensor can bemeasured independent of the oxidase target concentration. This may beaccomplished through physical separation on some nanometer, micro on mmscale of O₂ reference moieties from the enzyme-O₂ detection moieties toavoid cross talk. Such a reference measurement of O₂ would allowcorrections to be made to the glucose-specific signal from the oxidasesensing moieties.

In another embodiment, the polymer of the invention, preferably in theform of a tissue-integrating scaffold, may be a multi-analyte sensorwhere oxygen is one of two or more analytes detected and reported. Inthis embodiment, the polymer comprises a residue of compound of Formula1 and/or Formula 2 for detection of oxygen, and a second sensing moietyfor detection of another substance. Non-limiting examples of analytesthat may be detected by the sensing moieties include oxygen, reactiveoxygen species, glucose, lactate, pyruvate, cortisol, creatinine, urea,sodium, magnesium, calcium, potassium, vasopressin, hormones (e.g.,Luteinizing hormone), pH, cytokines, chemokines, eicosanoids, insulin,leptins, small molecule drugs, ethanol, myoglobin, nucleic acids (RNAs,DNAs), fragments, polypeptides, single amino acids and the like.

In another embodiment, the polymer of the invention, preferably in theform, of a tissue-integrating scaffold, may be a sensor where the oxygensignal, as detected by Formula 1 and/or Formula 2, is used as areference to correct or calibrate the signal for one or more otheranalytes. The oxygen signal may or may not be reported. It may be usedonly in internal algorithms to calibrate or correct the signal of theother analyte. The use of the oxygen signal as a reference in thisembodiment helps to overcome physiological fluctuations, which may alterthe analyte availability at the site of the sensor (e.g. blood flowvariations).

In still further embodiments, the sensing moieties, in addition to theresidue of compound of Formula 1 and/or Formula 2 comprise a secondluminescent analyte sensing moiety, and the residue of the compound ofFormula 1 and/or Formula 2 is used as a reference molecule. Thenon-oxygen sensing moieties may utilize analyte-specific moieties suchas competitive binding assays (e.g. a ligand receptor moiety and ananalyte analogue moiety such as Concanavalin A and dextran), reversibleluminescent binding molecules (e.g. boronic acid based sensing chemistryfor glucose detection), binding proteins such as glucose bindingproteins. To measure an analyte such as glucose in the tissue, thepolymer is illuminated from a patch reader on top of the skin above theimplant with 650 nm light at desired intervals over the long-term lifeof the implant (e.g., every 5-60 minutes over a period of 90 days ormore). The amount of luminescent signal (e.g., from a molecule such asAlexafluor 647) detected is proportional to the concentration of analyte(e.g. glucose) in the tissue. The amount of luminescent signal (e.g.from Formula 1 or Formula 2 molecule) detected is proportional to theconcentration of 02 in the tissue. The concentration of O₂ in the tissueis indicative of acute and or chronic physiological changes around thesensor, and may be used to correct or adjust the glucose signal or otheranalyte signal through a porportionality algorithm.

In another embodiment, internal reference control materials can beemployed that facilitate correcting for tissue optical variation. Thetissue-integrating implanted biosensor typically resides 3-4 mm underthe surface of the scan. It is well known that in skin excitation lightand emitted fluorescent light in the near infrared range are highlyscattered as the light traverses the tissue between the reader patch andthe implant. The extent of absorption and scattering is affected byphysical properties such as temperature or by tissue composition,including but not limited to variations in blood perfusion, hydration,and melanin concentration. Skin variations can occur between users orbetween different time points for a single patient, and these variationscan affect the fluorescence excitation and emissions signals causing inaccurate signals for the analyte-specific signal. Accordingly, aseparate fluorescence molecule with emission spectra distinguishablefrom the analyte-specific fluorescence can be immobilized into thescaffold. The fluorescence from the molecule can be measured separatelyfrom the analyte-specific fluorescence to measure a signal that informsabout variations in tissue composition. The dye selected is based onhaving a similar response to tissue variations as the analyte-specificdye. Formula 1 or Formula 2 may have the oxygen sensing capabilitiesgreatly reduced or eliminated, for example, by incorporation in anon-oxygen diffusive environment such as embedding in highly crosslinkedPAN or inside a silica shell. In this format, the dye molecules of thisinvention may serve as the stable internal reference control materialsdescribed above.

Tissue-integrating sensors comprised of one or more cylindrical shapedelements (e.g., fibers) eliminate or greatly reduce the foreign bodyresponse as compared to currently available implants. Moreover, theaverage diffusion distances from the capillary supply to all parts ofthe sensing media are comparable to native tissue, unlike other knownsensors.

It will be apparent that the overall dimensions of the sensing media(implantable sensor) will vary according to the subject and/or theanalyte(s) to be measured. Typically, the implant will be between about0.001 mm to 2 mm in thickness (or any value therebetween) and between 1mm and 1 cm in diameter (or an equivalent cross sectional area of anon-circular shape, for example length/width) and 15 mm in length orless, for example, a disk shaped sensor that is 2 mm or less thick and10 mm or less in diameter. In certain embodiments, the approximatesensor size is approximately 100-1000 microns in diameter and has thelength of between 0.25 mm and 10 mm. The size of the tissue-integratingsensing media in disk form is typically 2 mm or less thick and 10 mm orless in diameter.

Another aspect of the present invention is a tissue-integratingbiosensor system for semi-continuous, continuous and/or long-term usewithin a mammalian body.

One advantageous property of the polymers of the present invention istheir stability. In one aspect of the invention, the sensor is stable ina mammalian tissue for a long period of time, e.g., longer than a week,longer than a month, longer than 6 months. In one exemplary embodiment,as shown by the FIG. 2, the sensor is stable and produces a stablesignal when implanted into the rat skin for 170 days.

EXAMPLES

NMR spectroscopic data were recorded on a 300 MHz instrument at roomtemperature. NMR spectra were calibrated to the solvent signals ofdeuterated DMSO-d6 or CDCl3. The following abbreviations are used toindicate the signal multiplicity: s (singlet), d (doublet), t (triplet),q (quartet), br (broad), m (multiplet). Analytical HPLC-MS data, wererecorded on a HPLC system with a C18 reverse column coupled to anelectrospray ionization (ESI) spectrometer. 2-Aminoethyl methacrylatehydrochloride and tetraethylene glycol dimethacrylate were purchasedfrom Polysciences, Inc. All other chemicals were purchased from SigmaAldrich.

Example 1: Synthesis of a Polymerizable Near-IR Luminescent Dye

Scheme 1 describes the synthesis of one exemplary near-IR luminescentdye, Compound 2 (also referred to as Pd-BP):

Compound 3 was prepared as described in Niedermair et al, J. Inorg.Chem., 2010, 49, p. 9333. Briefly, to 90 mL of anhydrous THF was added1-nitrocyclohexenene (2.66 mL), ethyl isocyanoacetonitrile (2.6 mL), andDBU (3.53 mL). The reaction was refluxed at 70° C. under argon for 18hours. Brown precipitate formed as soon as heating began. THF wasevaporated, the residue was dissolved in methylene chloride, and theproduct was purified by flash chromatography on silica gel in methylenechloride. Product-containing fractions were evaporated under vacuum toremove most of the solvent, and to the residual solution hexanes wereadded to facilitate crystallization of the product. After 48 hr at 4°C., the precipitate was collected to by filtration to yield 2 g of theproduct as fine yellow needles. The mother liquor was partiallyevaporated to yield additional 1.4 g of the product; 75% total yield.

Compound 5: Compound 3 (1.40 g, 7.2 mmol) was suspended in 30 mL ofanhydrous ethylene glycol, and KOH pellets (0.73 g, 13.0 mmol) wereadded to the solution. The mixture was refluxed under argon for 1 hr.The resulting clear brown solution was cooled to 0° C., and 100 mL ofdichloromethane was added to the solution. Dichloromethane layer wasseparated, washed with water (2×100 mL), and brine (2×100 mL) and driedover anhydrous sodium sulfate. The product was purified by flashchromatography on silica gel in dichloromethane. Fractions containingthe fast-running component were pooled and diluted with dichloromethaneto 1000 mL. To the resulting solution was added methyl-4-formylbenzoate, under argon, the solution was stirred at room temperature for10 min, and BF₃.OEt₂ (0.19 mL, 1.3 mmol) was added. The mixture wasstirred for 2 hr, then 1.73 g (7.6 mmol) of DDQ was added, and themixture was allowed to stir overnight. The mixture was washedsequentially with 10% aq. Na₂CO₃, 1M HCl, and brine, then dried overanhydrous sodium sulfate. After purification by silica gelchromatography using stepwise gradient of MeOH in dichloromethane(0-2%), 430 mg (24%) of the product as green powder.

Compound 6: Compound 5 as a free base (0.43 g, 0.40 mmol) was dissolvedin 50 mL of benzonitrile. To the solution, PdCl2 was added under argon,and the mixture was refluxed for 10 min. The color of the solutionchanged from green to red. The mixture was cooled to room temperature,diluted with 200 mL of dichloromethane, and filtered through Celite.Dichloromethane was evaporated under vacuum, and benzonitrile wasdistilled off. The product was purified by flash chromatography onsilica get in dichloromethane, and the final purification was achievedby flash chromatography on silica gel in hexanes:ethyl acetate (1:1) toyield 0.109 mg (60%) of the product as a red powder.

Compound 7: Compound 6 (0.105 g, 0.09 mmol) was dissolved in 20 mL ofanhydrous THF, and DDQ (0.327 g, 1.44 mmol) was added to the solution.The mixture was refluxed for 20 min, and the reaction was stopped whenno starting material was detected in the mixture by TLC. THF was removedunder vacuum, the residue was diluted with dichloromethane and washedsequentially with 10% Na₂SO₄, water, and brine.

Compound 8: The ester 7 was hydrolyzed as described in Finikova et al.,J. Phys. Chem., 2007, 111, p. 6977. Briefly, 0.074 g (0.064 mmol) ofCompound 7 were dissolved in 110 mL of THF. To the solution, MeOH (10mL) was added, followed by a solution of 0.573 g of KOH in 2 mL of MeOH.Green precipitate formed in the solution, and the solution became almostcolorless. The precipitate was collected by centrifugation and dissolvedin 10 mL of water. The solution was acidified with 0.2 mL ofconcentrated HCl, and the resulting precipitate was collected bycentrifugation. Yield: 0.070 g (86%).

Compound 2: Compound 8, 30 (70 mg, 63.9 mmol) in DMF (10 mL) and CH₂Cl₂(10 mL) at 0° C. was added 1-hydroxybenzotriazole hydrate (43.17 mg,0.32 mmol), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride(61.25 mg, 0.32 mmol), and triethylamine (90 μL, 0.64 mmol). After 20min., 2-aminoethyl methacrylate hydrochloride (53.23 mg, 0.3195 mmol)was added, and the reaction was stirred for 16 h at room temperature.The CH₂Cl₂ was evaporated under reduced pressure, and ethylacetate/hexanes mixture was added to precipitate the crude product fromresidual DMF. The solvent was decanted, and the precipitated residue wasdissolved in CH₂Cl₂, washed sequentially with sat. NaHCO₃ and brine,dried over Na₂SO₄, filtered, and concentrated in vacuo. The crudeproduct was purified by flash chromatography on silica gel (gradient of0-4% methanol in CH₂Cl₂) to yield Compound 2 as a green powder (16 mg,16% yield). 1H NMR (300 MHz, CDCl₃) δ 8.40 (d, J=8.1 Hz, 8-11), 8.32 (d,J=8.1 Hz, 8H), 7.22 (br s, 8H), 7.10 (br s, 8H), 6.28 (s, 41H), 5.71 (s,4H), 4.61 (t, J=5.4 Hz, 8H), 4.03 (q, J=5.1 Hz, 8H), 2.06 (s, 12H).LC-MS (ESI): calcd for C₈₈H₇₃N₈O₁₂Pd: 1539.4403 [M+H]+, found 1539.4405[M+H]+, R_(t)=11.8 min.

Compound 9 was synthesized analogously to Compound 2 by reactingcommercially available tetracarboxyphenyl porphyrin with aminoethylmethacrylate in the presence of HOBt and EDC as shown in Scheme 2:

Example 2: Production of an Oxygen Sensing Media with Oxygen SensitiveLuminescent Dye Immobilized in a Tissue-Integrating Hydrogel Scaffold

The following describes one method for making a tissue-integratingsensor as described herein. This method involves the use ofnon-crosslinked PMMA templating microspheres and pHEMA as the scaffoldmaterial. The PMMA microsphere template was prepared using monodispersedPMMA spheres (20-100 um, preferably 80 urn) and placing the templatebeads between two glass slides with Teflon spacers. The sinteringprocess included sonicating for at least 10 minutes (one or more times)to closely pack the beads. Following sonication, the template is heatedto a sufficient temperature for a sufficient time to fuse the beads(typically, to 140-180° C. for 20-32 hours, for example, heat toapproximately 177° C. for 24 hours). For each lot of the beads, thetemperature and heating times are optimized.

The general preparation of an oxygen sensing poly(2-hydroxyethylmethacrylate) (pHEMA) scaffold was performed as follows: HEMA(2-hydroxyethyl methacrylate) (50 Wt %), TEGDMA(triethyleneglycol-dimethacrylate) (1 Wt %1), ethylene glycol (20 Wt %),water (25.5 W t %), the photoinitiator Irgacure 651 (0.5% vol/vol) and3% of Palladium-tetramethacrylate-benzoporphyrin (Compound 2,polymerizable O₂ sensitive dye) were mixed, yielding a finalconcentration of 1 mM Compound 2 in the polymer precursor solution.Polymer, solvents and sensing reagents were mixed to achievesufficiently high sensing chemistry concentration to measurably detect achange in signal through tissue.

The pre-mixed monomer solution was filled into the PMMA mold. Thesolution was placed under vacuum to remove any bubbles and to completelyinfiltrate the PMMA-mold. Polymerization was initiated by exposing themold to UV light (280-320 nm, 10-300 mW/cm2) for 5-10 minutes. Next, thePMMA microspheres were dissolved out of the resulting polymer byfrequent exchange of dichloromethane or other solvent system for 24-48hours using a Soxhlet extractor by manual volume changes.

The following describes preparation of the rod hydrogel sensors. 100 μLof a 10 mM solution of Compound 2 in DMSO, was added to a polymerprecursor solution [2-hydroxyethyl methacrylate (0.5 mL, 4.1 mmol),tetraethyleneglycol dimethacrylate (10 μL, 34 mmol), ethylene glycol(0.2 mL), water (185 μL) and 2,2-dimethoxy-2-phenylacetophenone (5 mg, 2mmol)], yielding a final concentration of 1 mM Compound 2. The dye andpolymer precursor mixture was injected into a poly(methyl methacrylate)(PMMA) bead-containing glass mold, as previously described by Marshall,A. J. et al. (Biomaterials with Tightly Controlled Pore Size thatPromote Vascular In-Growth. ACS Polymer Preprints 45, 100-101 (2004)).The mold was placed under vacuum to remove any bubbles and to ensurecomplete filling. Polymerization was initiated by exposing the mold toUV light (280-320 nm) using a Dymax 2000-EC Flood Curing System equippedwith a 400 Watt Mercury bulb for 2 minutes per side at a distance ofapproximately 6″. The glass plates were removed and the hydrogel wassoaked in 50 mL of CH₂Cl₂ (exchanged twice) with shaking for 24 hours toextract out the PMMA beads. The hydrogel was transferred into water andplaced under vacuum for 5 minutes to fully hydrate the porous scaffold.For implantation, the hydrogels were cut into rods (10 mm in length witha 750 μm×750 μm cross-section), disinfected by exposure to 70% ethanol,and then stored in sterile pH 7.4 PBS at 4° C. before use. Non-porous(i.e., solid) hydrogel sensors were prepared analogously but without theuse of templating beads.

Hydrogels comprising Glucose oxidase (GOx) were also prepared asdescribed above except GOx was also included in the polymerizationmixture used to prepare the scaffold (FIG. 5).

Example 3: Determination of Excitation and Emission Wavelengths ofCompound 2 Incorporated into a Hydrogel

The absorption and emission spectra of the dye-containing hydrogelsgenerated in Example 2 were measured in pH 7.4 PBS at ambient atmosphereusing a fluorescence plate reader (FIG. 1). The absorption spectracontained a Soret band at 445 um and a Q band at 633 nm. Excitation at633 nm gave an emission peak at 805 nm, thus confirming that Pd-BP(Compound 2) exhibits both absorption and emission in the NIR.

Example 4: Determination of Optimal Dye Concentration in Hydrogel

To determine the minimum dye concentration required to achieve a maximumintensity signal, a series of pHEMA hydrogels containing variousconcentrations of Pd-BP (Compound 2) were made. Solid and porous pHEMAhydrogels containing covalently-bound Pd-BP (Compound 2) at 0.01, 0.1,1, 2, and 3 mM dye concentrations were prepared. All gels were ˜1 mmthick; porous gels contained an average pore size of ˜70 hum. While inpH 7.4 PBS in ambient air, the fluorescence emission of each gel wasmeasured at 805 nm (633 nm excitation) using a fluorescence platereader. From these data, the optimal dye concentration was determined tobe 1 mM, since signal saturation was observed at higher concentrations.

Example 5: Characterization of Photo Bleaching of NIR Benzoporphyrin

Hydrogels containing covalently-bound Compound 2 were used inphotobleaching studies to determine the photostability of Compounds 2and 9. The hydrogels were tested in a custom-built flow-through systemintended to simulate physiological conditions (pH 7.4 PBS, 37° C., 21%02) while being illuminated by LED. The excitation light was directlydelivered to the bottom face of the gel samples via 1 mm diameter fiberoptic cables. Hydrogels containing Compound 9 were excited with a 525 nmLED source (power=127 mW/cm²) having a pulse duration (LED “on time”) of2 seconds and a pulse period of 5 seconds to achieve an overall dutycycle of 40%, while Compound 2 containing hydrogels were excited with a630 nm LED source (power=143 mW/cm²) with the same duty cycle. Theexperiment was run for 15 continuous hours under these conditions.However, less than 5% change in the lifetime signal of Compound 2 wasobserved. The resulting data from this experiment is used to estimatethe expected degree and rate of photobleaching which can occur duringlong-term in vivo use.

Gels containing the dye were extremely photostable when tested undersimulated use conditions (FIG. 6). These data indicate that measurementof the lifetime signal is a preferable strategy to achieve long-term (5months) stability in vivo. Photostability of the Pd-BP compound may befurther improved using techniques elsewhere disclosed, e.g. changing themetal core, or fluorinating or perfluorinating the base compound.

Example 6: Implantation

A tissue integrating sensor produced in rods that are 300-500 um indiameter and 5 mm long are placed in a 19-23 Gauge insertion needle,trochar, modified biopsy device or other devices engineered forinjection under the skin. The sensor is optionally dehydrated orcompressed before insertion to allow for the use of a smaller insertionneedle.

Upon insertion, skin is pinched up so that the insertion needle isplaced parallel to the surface of the skin up to 4 mm beneath thesurface. Fluid or a reverse displacement plunger (or trochar) is used toleave the sensor in the tissue as the syringe is withdrawn. Insertionsite may include any subcutaneous or dermal area, typically the abdomen,arm and thigh (FIG. 4). In research models, the dorsal skin, abdomen,hindlimb and brain (FIG. 3) have all been explored. The followingdescribes an example of hydrogel implantation, in-vivo fluorescentimaging, and data analysis in a rat model.

Hydrogel Implantation and In Vivo Fluorescent Imaging.

Hydrogel sensors (n=3 to 4 porous and n=3 to 4 solid), were injectedinto the subcutaneous tissue of 12 adult male CD rats (Charles RiverLabs, 150-250 g) for 1 week, 4 weeks, or 170 days. Rats wereanesthetized with 2-3% isoflurane (v/v in oxygen) during sensorinjection. Porous and solid hydrogel rods (10 mm long, 750 μm×750 μmcross-section) were loaded into 18 gauge needles and then inserted intothe dorsal subcutaneous space perpendicular to the midline. Sensors wereejected from the needle by inserting a stainless steel plunger throughthe cannula. Hydrogel sensors were implanted approximately 1.5 cm apart.Rats grew normally and showed no discomfort during the weeks followingthe sensor injection.

Oxygen sensors were fluorescently imaged once every 30 seconds in vivowith the IVIS Spectrum or Kinetic imaging system (Perkin Elmer, Waltham,Mass., USA). Rats were anesthetized at 2% isoflurane in 1.00 FIO2 for 30minutes prior to imaging. During in vivo imaging, the FIO2 was at leasttwice modulated down to 0.12 (v/v balance N2) for 5-10 minutes and thenreturned to 1.00 for 10-15 minutes. The relative response (intensity) ofeach sensor was quantified by identifying regions of interest (ROIs)surrounding the sensors and measuring the average radiant efficiency inthe ROI using the Living Image Software included with the IVIS System.

On the day of implantation, the Oxford Optronics OxyLite system was usedas a reference for tissue oxygenation. A needle-encased OxyLite probewas inserted subcutaneously in the dorsum of the rat on the day ofsensor injection (DO) and was allowed 10-15 minutes for the signal toreach a steady-state before data collection as described by Braun, et.al. (Comparison of tumor and normal tissue oxygen tension measurementsusing OxyLite or microelectrodes in rodents. Am J Physiol Heart CircPhysiol 280, H2533-2544 (2001)).

Data Analysis and Statistical Tests.

The data for each sensor, as defined by the ROI, was normalized to themaximum and minimum average radiant efficiency and inverted to have apositive correlation between the fluorescence data and tissueoxygenation. This normalization ensured that data for every sensor foreach separate experiment fell between 0 and 1, which were the maximumand minimum intensity of the sensor, respectively.

The sensors often did not reach a plateau during hypoxia testing becauseanimal health concerns necessitated the short exposure times (5-10 min).Therefore, to calculate the response time of the sensors, the time toachieve 90% of the fluorescent intensity change (T90%) during either the10 min hypoxic (FIO2=0.12) or the 15 min hyperoxic (FIO2==1.00) eventwas determined. The sensors were declared to have reached a steady stateif there was less than 10% of the total change over the last 3 minutesof the FIO2 change event. Data was tested for statistical significanceusing the non-parametric Wilcoxon rank-sum test (p<0.05).

Histological Analysis.

Rats were sacrificed and the sensors and surrounding tissue wereexplanted and frozen immediately in liquid nitrogen and stored at −80 C.Frozen tissue samples were cryosectioned at 10 μm thickness on a LeicaCM1850 cryostat and mounted on poly L-lysine coated glass slides.Sections were immunostained for rat CD31 (BD Biosciences, San Jose,Calif.). Briefly, slides were fixed in acetone for 20 min at roomtemperature, rinsed in 1×PBS, blocked with staining buffer (5% normaldonkey serum in 1×PBS) for 30 m, in, incubated with mouse-derived ratCD31 primary antibody at 1:200 in staining buffer for 1 h, and incubatedwith anti-mouse Alexa Fluor 488 (Jackson ImmunoResearch) for 30 min, andstained with Hoechst 33342 (Invitrogen) for 5 min at room temperature.Samples were fixed in 4% paraformaldehyde and imaged on the same day.Samples were fluorescently imaged using a Zeiss AxioSkop II+fluorescence microscope equipped with a 12 bit CCD camera (QImaging) andan automated scanning stage (Marzhauser) driven by a Ludl Mac5000driving unit (Ludl). An array of micrographs was acquired using a 5×objective (NA 0.25, Zeiss) and then stitched together to form a montageusing Metamorph software. Exposures were set at low illuminationintensities with 1×1 binning (pixel size of 1.36 μm×1.36 μm) and atypical acquisition period of 100 ms. The results of the experiment aredepicted in FIG. 11.

Example 7: Measurement

Data from the sensor is collected by a fluorescent reader placed on thesurface of skin directly above the sensor location, and the data isprocessed and displayed on a smart phone, other hand-held device,computer screen or other visualization format, for example usingcommercially available data display devices. Raw data is converted to ananalyte concentration or some non-quantitative representation of theanalyte concentration (e.g. high, low, within range). Values at anygiven point in time or trends (graphs over time) or summary statisticsover a period of time are provided. An indication of the quality of thedata is optionally provided. Hydrogels prepared from co-polymerizationof HEMA with NIR Pd-BP and green-ex Pd-TCPP were subcutaneouslyimplanted in a rat carcass, and their emission was measured (FIG. 2).Pd-BP was significantly brighter than Pd-TCPP due to the NIR excitationand emission wavelengths, which allow much greater light penetrationinto the skin, enabling deeper sensor placement. Deeper placement isdesirable for better immunological response, but was not possiblepreviously because the original green Pd-TCPP signal was largelyblocked, e.g., scattered and/or absorbed, by the skin. Only shallowdermal implants were possible. Additionally, Pd-BP hydrogel sensorsproduced bright detectable signal when implanted deep under a mouseskull (inside mouse brain).

Example 8: Stability of Sensors Implanted in Rat Skin

Oxygen sensors were implanted in rat skin and the intensity of theirsignal was monitored for 170 days. FIG. 4 shows fluorescence of thesensor implanted in a mouse skin for 170 days. Intensity varied as afunction of implantation depth (data was normalized to baselinefluorescence). Inhaled oxygen was modulated between 100% and 12% andimages were collected every 30 s in a Caliper IVIS (Spectrum, Ex=640 nm,Em=800 nm, 20 nm bandwidth). Regions of interest (ROIs) were drawnaround the sensors and the data plotted versus time (FIG. 6). This dataillustrate that the sensors made with the dyes of the present inventionmaintain function for many months in vivo. Additionally, thetissue-integrating sensor was compared to a solid sensor. Thetissue-integrating sensor produced a faster kinetic response to changesin oxygen levels than the solid sensor, which illustrates anotheradvantageous property of the tissue-integrating sensor.

Example 9: In-Vitro Oxygen Detection in Low Oxygen Concentrations

To characterize oxygen sensitivity of Pd-BP, the intensity andluminescence lifetime of the dye in a porous HEMA hydrogel at various O₂levels (0%, 12%, and 20% O2) was measured (FIG. 8). The hydrogels weretested in a custom-built flow-through system (pH 7.4 PBS, 37° C.) whilebeing monitored with the TauTheta fiber-optic instrument. The dye showedgood reversibility, as well as good O₂ sensitivity as indicated by theStern-Volmer plot.

Example 10: Preparation and Characterization of Glucose Sensor

Glucose oxidase (GOx) was entrapped in a pHEMA hydrogel containingcovalently bound Pd-BP as described above. The porous morphology of theresulting sensor was confirmed with SEM (FIG. 5). The GOx-Pd-BP sensorswere tested for glucose response in a flow-through system (PBS, 37° C.).The luminescence intensity and lifetime of Pd-BP within the gel weremonitored during a series of glucose excursions spanning thephysiological range (FIG. 9). The slight dip in intensity and lifetimeduring the plateaus (where glucose concentration was held constant) aredue to consumption of glucose within the test reservoir by GOx.

Example 11: Implantation of O2 Sensor into Pig Skin

Explant specimens were obtained from acute pig experiment during whichtime the O₂ sensors prepared from the polymers comprising Compound 2were injected into a pig. Sensor signals were obtained. Fluorescencelifetime and intensity measurements were collected. After sensor signalmeasurements were obtained, the pig was sacrificed and specimens werefixed in 10% Formalin and stained with Hematoxylin and Eosin (H&E).Images and depth measurements were obtained using a Nikon microscope at40× magnification and the Infinity 1 microscopy camera and software(Version 6.1.0, Luminera Corp.) Sequential overlapping images wereobtained to create the final composite images. FIG. 10 depicts a sensorthat was found to have been implanted at 8 mm in depth under the surfaceof the skin. FIG. 10 shows that modulating sensor signal was stilldetectable at the depth of sensor implantation of 8 mm under the surfaceof the skin.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

All patents, patent applications and publications mentioned herein arehereby incorporated by reference in their entirety. Although disclosurehas been provided in some detail by way of illustration and example forthe purposes of clarity of understanding, it will be apparent to thoseskilled in the art that various changes and modifications can bepracticed without departing from the spirit or scope of the disclosure.Accordingly, the foregoing descriptions and examples should not beconstrued as limiting.

What is claimed is:
 1. A sensor for detecting an analyte comprising apolymer, wherein said polymer comprises one or more residues of aluminescent dye, and wherein the luminescent dye is a photostableporphyrin dye and has excitation and emission spectra in the NIR opticalwindow of a mammalian skin; wherein the luminescent dye is a compoundhaving the formula:

wherein M is H, Pd, Zn, Pt, Gd or Yb; each R¹ is same or different and,independently, C(O)X—(CH₂)_(n)—YC(O)C(R⁴)CH₂ orC(O)X—(CH₂CH₂O)_(m)CH₂CH₂—YC(O)C(R⁴)CH₂; R⁷ isC(O)X—(CH₂)_(n)—YC(O)C(R⁴)CH₂ orC(O)X—(CH₂CH₂O)_(m)CH₂CH₂—YC(O)C(R⁴)CH₂; R² and R³ are fused, in eachcase, to form a cycloalkenyl, aryl, or heteroaryl group; X is O or NR⁵;Y is O or NH; R⁴ is H or C₁-C₄ alkyl; R⁵ is C₁-C₄ alkyl; each R⁶ is sameor different and, independently, H or F; n is 1-10; and m is 1-300. 2.The sensor of claim 1 wherein the luminescent dye has an absorptionmaximum between 500 nm and 900 nm and an emission maximum between 600 nmand 1000 nm.
 3. The sensor of claim 1 wherein the residue of theluminescent dye is present at a concentration from about 0.01 mM toabout 5 mM.
 4. The sensor of claim 1 wherein the residue of theluminescent dye is present at the concentration of about 1 mM.
 5. Thesensor of claim 1 wherein the polymer further comprises the residues ofHEMA.
 6. The sensor of claim 1 wherein the polymer is a hydrogel.
 7. Thesensor of claim 1 wherein the analyte is oxygen.
 8. The sensor of claim1 wherein the sensor is tissue-integrating.
 9. The sensor of claim 1wherein the sensor generates detectable luminescent signal when placedunder the skin of a mammalian subject.
 10. The sensor of claim 9 whereinthe sensor generates detectable luminescent signal when placed up toabout 5 mm deep under the skin of a mammalian subject.
 11. The sensor ofclaim 9 wherein the sensor generates detectable luminescent signal whenplaced more than 1 mm deep under the skin of a mammalian subject. 12.The sensor of claim 9 wherein the mammalian subject is a human.
 13. Thesensor of claim 9 wherein the sensor is stable in a mammalian tissue forlonger than 1 week.
 14. The sensor of claim 1 further comprising anoxidase.
 15. The sensor of claim 14, wherein the oxidase is a glucoseoxidase, ethanol oxidase, lactate oxidase, pyruvate oxidase, bilirubinoxidase, or histamine oxidase.
 16. The sensor of claim 14 wherein theanalyte is oxygen consumed by the oxidase.
 17. The sensor of claim 16,wherein the oxidase is a glucose oxidase, ethanol oxidase, lactateoxidase, pyruvate oxidase, bilirubin oxidase, or histamine oxidase. 18.The sensor of claim 17 wherein the sensor is tissue-integrating.
 19. Asensor for detecting an analyte comprising a polymer, wherein saidpolymer comprises one or more residues of a luminescent dye, and whereinthe luminescent dye is a photostable porphyrin dye and has excitationand emission spectra in the NIR optical window of a mammalian skin;wherein the luminescent dye is a compound having the formula:

wherein M is H, Pd, Zn, Pt, Gd or Yb; each R¹ is same or different and,independently, C(O)X—(CH₂)_(n)—YC(O)C(R⁴)CH₂ orC(O)X—(CH₂CH₂O)_(m)CH₂CH₂—YC(O)C(R⁴)CH₂; R⁷ isC(O)X—(CH₂)_(n)—YC(O)C(R⁴)CH₂ orC(O)X—(CH₂CH₂O)_(m)CH₂CH₂—YC(O)C(R⁴)CH₂; R² and R³ are fused, in eachcase, to form a cycloalkenyl, aryl, or heteroaryl group; X is O; Y is Oor NH; R⁴ is H or C₁-C₄ alkyl; each R⁶ is same or different and,independently, H or F; n is 1-10; and m is 1-300.
 20. The sensor ofclaim 19 wherein the luminescent dye has an absorption maximum between500 nm and 900 nm and an emission maximum between 600 nm and 1000 nm.21. The sensor of claim 19 wherein the residue of the luminescent dye ispresent at a concentration from about 0.01 mM to about 5 mM.
 22. Thesensor of claim 21 wherein the residue of the luminescent dye is presentat the concentration of about 1 mM.
 23. The sensor of claim 19 whereinthe polymer further comprises the residues of HEMA.
 24. The sensor ofclaim 19 wherein the polymer is a hydrogel.
 25. The sensor of claim 19wherein the analyte is oxygen.
 26. The sensor of claim 19 wherein thesensor is tissue-integrating.
 27. The sensor of claim 26 wherein thesensor generates detectable luminescent signal when placed under theskin of a mammalian subject.
 28. The sensor of claim 27 wherein thesensor generates detectable luminescent signal when placed up to about 5mm deep under the skin of a mammalian subject.
 29. The sensor of claim27 wherein the sensor generates detectable luminescent signal whenplaced more than 1 mm deep under the skin of a mammalian subject. 30.The sensor of claim 27 wherein the mammalian subject is a human.
 31. Thesensor of claim 27 wherein the sensor is stable in a mammalian tissuefor longer than 1 week.
 32. The sensor of claim 19 further comprising anoxidase.
 33. The sensor of claim 32, wherein the oxidase is a glucoseoxidase, ethanol oxidase, lactate oxidase, pyruvate oxidase, bilirubinoxidase, or histamine oxidase.
 34. The sensor of claim 32 wherein theanalyte is oxygen consumed by the oxidase.
 35. The sensor of claim 34,wherein the oxidase is a glucose oxidase, ethanol oxidase, lactateoxidase, pyruvate oxidase, bilirubin oxidase, or histamine oxidase. 36.The sensor of claim 35 wherein the sensor is tissue-integrating.